The goal of the research described in this proposal is to increase our basic understanding of the intramolecular interactions and protein-solvent interactions that determine the three dimensional structure of proteins in solution. Such information is necessary to understand protein folding and needed to predict the conformation of a protein from its amino acid sequence. Ultimately, this knowledge will be useful in designing proteins to perform specific functions. The specific aims of the proposed research are to examine the conformations, unfolding processes, and hydration of protein ions and peptide ions in the gas phase. The advantage of gas phase studies is that it is possible to separate the intramolecular interactions and protein-solvent interactions, and study them individually. In solution, there is a complex mixture of interactions and they cannot be separated. The approach employed here is unconventional, but the fundamental molecular interactions responsible for the three-dimensional structure of a protein are the same in the gas phase and solution phase and so the results of the gas-phase studies can be related back to the solution environment. The proposed experiments will exploit the recent developments in biomedical mass spectrometry that make it possible to produce dehydrated protein ions in the gas phase. Studies will be performed for a number of proteins and synthetic peptides, selected to examine key issues, such as the intrinsic alpha-helix propensity of the different amino acids in the gas phase. The gas phase conformations will be examined by ion mobility measurements. Comparison of the experimental results with the predictions of molecular dynamics simulations will provide insight into the unfolding and refolding processes, and test the theoretical methods used to model proteins. Information about protein-solvent interactions will be obtained from experiments where a known number of water molecules are added to the dehydrated protein ions in the gas phase. Measurements of the enthalpy and entropy changes associated with adsorbing individual water molecules will be performed, along with an examination of how the adsorbed water molecules modify the structure of the protein.